The Functional Relationship between the Cdc50p-Drs2p Putative Aminophospholipid Translocase and the Arf GAP Gcs1p in Vesicle Formation in the Retrieval Pathway from Yeast Early Endosomes to the TGN

Sakane, Hiroshi; Yamamoto, Takaharu; Tanaka, Kazuma

doi:10.1247/csf.06021

Cited by 40 publications

(40 citation statements)

References 88 publications

(152 reference statements)

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“…1B). In the Cdc50p-depleted cells in which Cdc50p was partially depleted for 12 h in the presence of glucose, Dnf2p-GFP was internally accumulated in some cells, but most of the cells still exhibited polarized Dnf2p-GFP (90%, n ϭ 122), as reported previously for GFP-Snc1p (18). In contrast, in the Cdc50p-depleted neo1-101 mutant, only 8% (n ϭ 127) of the cells exhibited polarized Dnf2p-GFP, and the remaining 92% accumulated Dnf2p-GFP in internal structures that seemed to be early endosome-derived abnormal membranes.…”

Section: Cho1-gfp Ura3supporting

confidence: 84%

“…Cdc50p-Drs2p, which is mainly localized to early endosome/ TGN membranes, is implicated in the formation of clathrincoated vesicles from these membranes (16)(17)(18)(19). Because clathrin has no intrinsic lipid-binding ability, it is linked to membranes by adaptors, which bind to lipids and/or the cytoplasmic domains of cargo proteins (20).…”

mentioning

confidence: 99%

“…Because clathrin has no intrinsic lipid-binding ability, it is linked to membranes by adaptors, which bind to lipids and/or the cytoplasmic domains of cargo proteins (20). The AP-1 clathrin adaptor has been suggested to function downstream of Cdc50p-Drs2p (18,19), but the underlying mechanisms are unknown.…”

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confidence: 99%

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Role of Phosphatidylserine in Phospholipid Flippase-Mediated Vesicle Transport in Saccharomyces cerevisiae

2014

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Phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. The genome of budding yeast encodes four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), which associate with the Cdc50 family noncatalytic subunit, and one monomeric flippase Neo1p. Flippases have been implicated in the formation of transport vesicles, but the underlying mechanisms are largely unknown. We show here that overexpression of the phosphatidylserine synthase gene CHO1 suppresses defects in the endocytic recycling pathway in flippase mutants. This suppression seems to be mediated by increased cellular phosphatidylserine. Two models can be envisioned for the suppression mechanism: (i) phosphatidylserine in the cytoplasmic leaflet recruits proteins for vesicle formation with its negative charge, and (ii) phosphatidylserine flipping to the cytoplasmic leaflet induces membrane curvature that supports vesicle formation. In a mutant depleted for flippases, a phosphatidylserine probe GFP-Lact-C2 was still localized to endosomal membranes, suggesting that the mere presence of phosphatidylserine in the cytoplasmic leaflet is not enough for vesicle formation. The CHO1 overexpression did not suppress the growth defect in a mutant depleted or mutated for all flippases, suggesting that the suppression was dependent on flippase-mediated phospholipid flipping. Endocytic recycling was not blocked in a mutant lacking phosphatidylserine or depleted in phosphatidylethanolamine, suggesting that a specific phospholipid is not required for vesicle formation. These results suggest that flippase-dependent vesicle formation is mediated by phospholipid flipping, not by flipped phospholipids.

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Section: Cho1-gfp Ura3supporting

confidence: 84%

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confidence: 99%

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Role of Phosphatidylserine in Phospholipid Flippase-Mediated Vesicle Transport in Saccharomyces cerevisiae

2014

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“…We originally linked Drs2p to clathrin-mediated transport through the recovery of several drs2 alleles in an arf1 synthetic lethal screen and the observation that drs2 is also synthetically lethal with clathrin heavy-chain temperature-sensitive alleles but not with mutations in COPI or COPII subunits (Chen et al, 1999). Subsequently, genetic interactions were discovered between Drs2p and ARF guanine nucleotide exchange factors (Gea1p and Gea2p), an ARF guanine nucleotide activating protein (Gcs1p), GGAs, Ypt31p (a rab protein), TRAPPII subunits, and a phosphatidylinositol 4-kinase (Pik1p; Chantalat et al, 2004;Sciorra et al, 2005;Sakane et al, 2006). In addition, the Drs2-Cdc50 complex physically interacts with Gea2p, AP-1, and Rcy1p, providing a direct link to ARF/AP-1/clathrin pathways as well as the Rcy1-dependent early endosometo-TGN recycling pathway (this work and Chantalat et al, 2004;Furuta et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

“…This Drs2-dependent flippase activity also appears to contribute to the asymmetric distribution of endogenous PS and PE to the cytosolic leaflet as drs2⌬ mutants exhibit a loss of PS and PE asymmetry (Pomorski et al, 2003;Chen et al, 2006). P4-ATPases also play essential roles in protein transport in the secretory and endocytic pathways (Chen et al, 1999;Hua et al, 2002;Hua and Graham, 2003;Pomorski et al, 2003;Sakane et al, 2006;Furuta et al, 2007). For example, inactivation of Drs2-ts in vivo rapidly blocks formation of a clathrin-dependent class of post-Golgi vesicles carrying exocytic cargo (Gall et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

P4-ATPase Requirement for AP-1/Clathrin Function in Protein Transport from the trans-Golgi Network and Early Endosomes

Liu

Surendhran

Nothwehr

et al. 2008

MBoC

110

158

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Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2⌬ cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN. INTRODUCTIONClathrin mediates protein transport between the plasma membrane, endosomes, and TGN through association with pathway-specific adaptors that link integral membrane cargo proteins to the clathrin lattice during vesicle formation. In mammalian cells, the ubiquitously expressed heterotetrameric AP-1A clathrin adaptor, composed of ␥, ␤1, 1A, and 1-adaptins, appears to mediate both anterograde transport of proteins from the TGN to early endosomes, as well as the retrograde trip back to the TGN (Hinners and Tooze, 2003;Robinson, 2004). For its role in anterograde transport of lysosomal enzymes, AP-1A collaborates with monomeric adaptors called GGAs (Golgi localized, ␥-earcontaining, Arf-binding proteins) to recruit the mannose-6-phosphate receptor into clathrin-coated vesicles (CCVs; Doray et al., 2002). The AP-1B complex, which differs from AP-1A by an alternate 1B subunit, has a functionally distinct role in sorting cargo from the TGN to the basolateral membrane of polarized epithelial cells (Folsch et al., 1999). In budding yeast, there is evidence that AP-1 mediates early endosome to TGN retrograde transport (Valdivia et al., 2002;Foote and Nothwehr, 2006), but no anterograde role for AP-1 has yet been described. The yeast GGAs appear to function independently of AP-1 to mediate delivery of cargo from the TGN to the late endosome (Black and Pelham, 2000). Deletion of genes encoding AP-1 subunits or GGAs has little consequence to growth of yeast. However, the combined deletion of AP-1 and GGA causes a severe growth defect, suggesting that these two adaptors mediate parallel transport pathways and that yeast cannot tolerate loss of both pathways (Costaguta et al., 2001;Hirst et al., 2001).The mechanism of AP-1/clathrin-coated vesicle ...

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Coupling Drs2p to Phospholipid Translocation, Membrane Asymmetry, and Vesicle Budding

Zhou

Natarajan

Muthusamy

et al. 2011

Transmembrane Dynamics of Lipids

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The Functional Relationship between the Cdc50p-Drs2p Putative Aminophospholipid Translocase and the Arf GAP Gcs1p in Vesicle Formation in the Retrieval Pathway from Yeast Early Endosomes to the TGN

Cited by 40 publications

References 88 publications

Role of Phosphatidylserine in Phospholipid Flippase-Mediated Vesicle Transport in Saccharomyces cerevisiae

Role of Phosphatidylserine in Phospholipid Flippase-Mediated Vesicle Transport in Saccharomyces cerevisiae

P4-ATPase Requirement for AP-1/Clathrin Function in Protein Transport from the trans-Golgi Network and Early Endosomes

Coupling Drs2p to Phospholipid Translocation, Membrane Asymmetry, and Vesicle Budding

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